Large conductance Ca2+-activated K+ channel (BKCa) α-subunit splice variants in resistance arteries from rat cerebral and skeletal muscle vasculature.

Zahra Nourian,Min Li,Michael A Hill,Jonathan H Jaggar,Andrew P Braun,M Dennis Leo,Yu Huang

doi:10.1371/journal.pone.0098863

Zahra Nourian, Min Li + Show 5 more

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https://doi.org/10.1371/journal.pone.0098863

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Abstract

Previous studies report functional differences in large conductance Ca2+ activated-K+ channels (BKCa) of smooth muscle cells (VSMC) from rat cerebral and cremaster muscle resistance arteries. The present studies aimed to determine if this complexity in BKCa activity may, in part, be due to splice variants in the pore-forming α-subunit. BKCa variants in the intracellular C terminus of the α-subunit, and their relative expression to total α-subunit, were examined by qPCR. Sequencing of RT-PCR products showed two α-subunit variants, ZERO and STREX, to be identical in cremaster and cerebral arteries. Levels of STREX mRNA expression were, however, significantly higher in cremaster VSMCs (28.9±4.2% of total α-BKCa) compared with cerebral vessels (16.5±0.9%). Further, a low level of BKCa SS4 α-subunit variant was seen in cerebral arteries, while undetectable in cremaster arteries. Protein biotinylation assays, in expression systems and arterial preparations, were used to determine whether differences in splice variant mRNA expression affect surface membrane/cytosolic location of the channel. In AD-293 and CHO-K1 cells, rat STREX was more likely to be located at the plasma membrane compared to ZERO, although the great majority of channel protein was in the membrane in both cases. Co-expression of β1-BKCa subunit with STREX or ZERO did not influence the dominant membrane expression of α-BKCa subunits, whereas in the absence of α-BKCa, a significant proportion of β1-subunit remained cytosolic. Biotinylation assays of cremaster and cerebral arteries showed that differences in STREX/ZERO expression do not alter membrane/cytosolic distribution of the channel under basal conditions. These data, however, revealed that the amount of α-BKCa in cerebral arteries is approximately 20X higher than in cremaster vessels. Thus, the data support the major functional differences in BKCa activity in cremaster, as compared to cerebral VSMCs, being related to total α-BKCa expression, regardless of differences in splice variant expression.

Highlights

Potassium channels play an important role in the regulation of VSMC membrane potential and contractile activity
The functional BKCa channel exists as a tetramer of a-subunits forming the ion channel pore together with tissue specific auxiliary b-subunits (b1–b4) which are typically present in a 1:1 stoichiometry [7]
We showed that BKCa from cremaster VSMCs exhibit a decreased Ca2+ sensitivity and suggested that this may, in part, be due to a decrease in the amount of the b1 regulatory subunit

Summary

Introduction

Potassium channels play an important role in the regulation of VSMC membrane potential and contractile activity. The BKCa a-subunit consists of seven transmembrane spanning domains (S0–S6) including the extracellular N-terminus, P-loop between S5 and S6 domains, and a large intracellular C terminus containing a number of regulatory sites including the regulators of conductance for K+ (RCK 1 and 2) and 2–3 Ca2+ binding sites. BKCa channels appear to achieve part of their functional diversity through alternative premRNA splicing of the KCNMA1 gene [9,10]. Alternative splicing can modify the functional properties of BKCa channels, including Ca2+ and voltage sensitivity, cell surface expression, and regulation by diverse intracellular signaling pathways. It has been shown that the STREX exon (an insertion of 58 amino acids in the C-terminal splice site 2 of the a-subunit protein) confers distinct functional phenotypes onto BKCa channels, such as altered Ca2+ sensitivity and changing responsiveness of channels to cAMP

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